Approximately 15% diabetics will have an ulcer in their lifetime. Annual, population-based incidence of 1% to 3.6% among people with type 1 or type 2 diabetes. By 2030, approximately 13 million people will suffer a diabetic foot ulcer each year. Foot ulcers precede more than 84% of non-traumatic lower limb amputations. In addition, the 3-year mortality after a first amputation has been estimated as high as 20-50%. The average cost of healing a single ulcer is $8,000, that of an infected ulcer is $17,000, and that of a major amputation is $45,000. The excess cost attributed to foot ulcers and their sequelae averaged $27,987 per patient for a 2-year period following ulcer presentation (American Diabetes Association; US Department of Health and Human Services; Diabetes Care; Journal of Clinical investigations; JAMA 293:217-228, 2005; Diabetes Care 22:157-162, 1999).
Free blood flow to a site of injury provides access for signaling molecules and nutrients. Regulation of proliferation and remodelling of collagen formation requires coordination of molecular processes (e.g. protease and protease inhibitor balance). Immune and inflammatory response ensure bacteria and debris are phagocytosed and removed prevent infection (J Invest Dermatol. 2007 May; 127(5):1018-29). If untreated, a diabetic ulcer can progress from a small irritated but unbroken skin patch to a potentially life-threatening wound involving extensive tissue death and infection. Treatment of the diabetic skin ulcer may include drying out the wound, debriding (excising) the dead tissue, and administering systemic antibiotics. (American College of Foot and Ankle Surgeons; Medscape website; Clinical Diabetes Spring 2009 vol. 27 no. 2 52-58).
In regard to the use of platelet lysate in cosmetics it is well known that healthy skin requires tight coordination at the physiological, cellular and molecular levels. Three critical functions required for sustained healthy skin include functioning connective tissue cells, intact skin extracellular matrix (ECM), and tight molecular regulation of the proliferation and remodeling process.
The process to form new tissue requires the action and balance of a number of factors including proteases, tissue inhibitors, and pro- and anti-inflammatory molecules. Coordinated regulation of these factors ensures permanent collagen tissue is being generated while the temporary scaffold is degraded.
Functioning Connective Tissue Cells
Cells within the skin, such as fibroblasts and keratinocytes, are required to synthesize, maintain and provide the extracellular matrix (ECM) that functions as the structural framework of the skin. It is especially important for fibroblasts, which produce the majority of collagen in the skin, to remain healthy and elastic [1]. Platelet and growth factor based solutions enhance wound healing by increasing local concentration of beneficial factors [2, 3]. Current therapies are invasive or minimally accelerate the process of wound healing. An advanced dressing such as hydrocolloid gel have limited improvement over traditional non-adherent gauze in the treatment of diabetic ulcers [6]. Technological breakthroughs in wound healing have also been limited over the last 15 years, with little improvement regarding patient quality-adjusted life year (QALY) outcomes [7].
In healthy skin, intact type I collagen fibrils in the dermis provide mechanical stability and attachment sites for fibroblasts. Receptors (integrins) on the surface of fibroblasts attach to collagen (and other proteins in the dermal extracellular matrix). Cytoskeletal machinery (actin-myosin microfilaments, not shown) within fibroblasts pulls on the intact collagen matrix, which in turn offers mechanical resistance. Dynamic mechanical tension that is created promotes assembly of intracellular scaffolding (microtubules/intermediate filaments, not shown), which pushes outward to cause fibroblasts to stretch. This stretch is required for fibroblasts to produce normal levels of collagen and proteases.
Intact Skin Extracellular Matrix (ECM)
A healthy balance of intact collagen (the most abundant protein in ECM), elastin, hyaluronic acid, proteoglycans, fibronectin, and laminin are necessary for healthy, wrinkle-free skin. These components create a scaffold to support the skin and give it an evenly distributed texture [8].
Tight Molecular Regulation of the Proliferation and Remodelling Process.
Forming new tissue requires the action and balance of a number of factors including proteases, tissue inhibitors, and pro- and anti-inflammatory molecules. Coordinated regulation of these factors ensures permanent collagen tissue is being generated; damaged molecules are cleaved and cleared, and overall health of the connective tissue [9].
All fibrillar collagens consist of three polypeptide chains wound around each other in a triple helical configuration. The soluble triple helix, which is termed procollagen, is assembled inside fibroblasts. Procollagen is secreted from fibroblasts, and the peptide ends are removed by two enzymes in the extracellular space [10]. Removal of the ends produces collagen, which spontaneously assembles (i.e., matures) into large fibres that are enzymatically cross-linked. This cross-linking is necessary for normal structural support [11]. Type I collagen undergoes natural breakdown by enzymatic degradation; however, this degradation in human skin is exceedingly slow [12]. Humans express only four enzymes that are capable of initiating breakdown of type I collagen [13].
These collagenases are members of a family of matrix protein-degrading enzymes, referred to as matrix metalloproteinases (MMPs) [14]. MMPs are responsible for physiological degradation of various extracellular matrix proteins [12]. Of the four collagenases that are expressed in humans, only interstitial collagenase (MMP-1) is involved in normal turnover of skin collagen [14]. In healthy young skin, MMP-1 expression is exceedingly low, near the limit of detection by the most sensitive measurement methods.
Once cleaved by MMP-1, collagen unravels, then un-raveled collagen, called gelatin, then undergoes further degradation by other members of the MMP family, called gelatinases. These gelatinases are also expressed at very low levels in normal skin [14, 15]. In addition, skin expresses natural inhibitors of these MMPs. These tissue inhibitors of matrix metalloproteinases (TIMPs) further act to retard collagen breakdown. Thus, type I collagen in human skin is very stable, requiring approximately 30 years on average to undergo replacement [1, 12].
Wrinkles
Wrinkles are caused by habitual facial expressions, aging, sun damage, smoking, poor hydration and various other factors. Under stress, fibroblasts produce less ECM molecules and more molecules which break down the existing matrix. This leads to further malfunctioning of the connective tissue cells in a feedback loop.
Malfunctioning of Connective Tissue Cells
A delicate relationship exists between mechanical tension, collagen synthesis and collagen fragmentation by collagenase (COLase) in human skin. In aged human skin, attachments of fibroblasts to integrins are lost and fragmented collagen fibrils fail to provide sufficient mechanical stability to maintain normal mechanical tension. Reduced mechanical tension causes fibroblasts to collapse, and collapsed fibroblasts produce less procollagen and more collagenase (COLase). Reduced collagen production and increased collagenase-catalysed collagen fragmentation result in further reduction of mechanical tension, thereby causing continual loss of collagen [1].
Breakdown of the Existing ECM
The existing ECM molecules, especially collagen fibres, are broken down through physical, chemical, or proteolytic damage, leaving collagen and other molecular fragments throughout the ECM.
The slow rate of type I collagen turnover allows accumulation of age-dependent modifications that impair its functions. These alterations include formation of new cross-links derived from sugars [14]. Importantly, these crosslinks are not able to be efficiently broken down and removed during the slow normal process of MMP-mediated turnover, causing accumulation of fragmented collagen within the extracellular matrix as skin ages [15, 16]. Cross-links prevent complete removal of collagen fragments. The fragments cannot be repaired or incorporated into newly made collagen fibrils, and therefore cause defects in the three dimensional collagen matrixes. These defects impair the structural and mechanical integrity of the dermis and thereby deleteriously alter its function. Accumulation of fragmented collagen lies at the heart of age-related changes in the appearance of human skin [1].
Deregulated Molecular Signaling
Collapsed fibroblasts and degraded ECM disproportionately increase the concentration of matrix metalloproteases to tissue inhibitors of metalloproteases (TIMP) at the wrinkle site, impeding tissue regeneration [1, 10-20].
Reactive oxygen species (ROS), a by-product of both environmentally induced and intrinsic aging, cause a cascade of biochemical reactions within the skin, which results in the production of matrix metalloproteinases (MMPs) and proinflammatory cytokines. MMPs, secreted by fibroblasts and keratinocytes, decrease collagen formation and enhance collagen degradation, contributing to the breakdown of the dermal matrix [19].
Current Wrinkle Therapies
Current treatments on the market fail, they do not recreate youthful appearance with lasting results. They aim to treat the symptoms of wrinkles without addressing the root causes. Toxins temporarily relax muscles in the face that stretch the skin, but does not repair the underlying extracellular matrix. Botox® and Dysport® can result in ‘frozen face,’ with problems swallowing, speaking, and breathing. Fillers (such as hyaluronic acid, collagen, poly-L-lactic acid, and hydroxylapatite) ‘fill the gaps’ for a short period, but does not restore skin to normal physiology or prevent the degenerative cycle.
Technological breakthroughs in cosmetics have been limited over the last 30 years, with sporadic advancements over the last decade. First and second generation injectable therapies have a quick time to onset, but a limited duration of effect.
Platelet Lysate Therapies
There are several long standing issues affecting the wide spread adaptation and use of platelet lysate (PL) in the clinic. The main issues have been, but are not limited to the following: donor derived samples run the risk of contamination during processing; platelet quality varies from patient to patient in terms of count and ability to secrete beneficial growth factors making the preparations inconsistent; there is a need for platelet quantitation in physician office kits; therapeutic preparation introduces high patient-by-patient variability affecting efficacy due to intrinsic differences in platelet count; current methods are inconvenient, clinicians must centrifuge blood to isolate platelets from blood, and the variability of this process, which can last between 25-30 minutes, again introduces errors; and the current processes are not cost-effective with an estimated cost of $130 per treatment (Plateltex). The invention is a non-obvious solution to these long standing and persistent problems.